| [Background]Chemotherapy is one of the most effective tools in breast cancer treatment. However, the emergence of MDR to a series of clinical chemotherapeutics with different structures or different target sites, severely blocks the success of breast cancer chemotherapy. The well recognized mechanism of classical MDR is the significant overexpression of human MDR1 gene encoding MDR1/p-gp.The MDR1/p-gp acts as an efflux pump in the cell surface. Intracellular anti-cancer drugs increasingly flows from the cells through the efflux pump, leading to lower drug concentration, thereby making the cancer cells resistant to chemotherapeutic drugs, such as doxorubicin and paclitaxel.Nowadays, modulators or inhibitors of P-gp are being used to reverse MDR. However, the currently available P-gp inhibitors or modulators have not been able to completely restore MDR mainly because of their side effects. Therefore, targeting genes that are extensively involved in the development of MDR provides a possible way out in the breast cancer chemotherapy.Nonvisual β-arrestins, β-arrestinl and β-arrestin 2, are ubiquitously localized in the cytoplasm and plasma membrane. They were firstly discovered as scaffold proteins involved in G protein-coupled receptors desensitization, sequestration, and internalization. Recently, growing researches indicates that β-arrestin2 plays an important role in many pathological progresses. Especially, it is widely involved in many cancer developmental signaling pathways that contribute to malignant tumor progression. In addition, increasing evidences have suggested that β-arrestin2 plays a potential role in tumor viability and metastasis. Mistre Alemayehu et al. discovered that β-arrestin2 could directly regulate the LPA1-induced migration and invasion in breast cancer MDA-MB-231 cell line. Buchanan et al. also reported that, P-arrestin 2 mediated metastasis in mouse colorectal tumor entity. Besides, Zhao et al. demonstrated that β-arrestin 2 has anti-apoptosis function in human breast cancer cells mediated through caspase-8 pathways. Just recently, we found that β-arrestin 2 is relative with MDRl/P-gp mediated chemotherapy resistance in breast cancer.In our experimental work, we unintentionally found that the expression of β-arrestin2 was closely related with the MDRl/P-gp expression in our clinical samples of breast carcinoma. We also examined the relationships between β-arrestin2 and MDR1/p-gp in three breast cancer cell lines and inferred that β-arrestin2 has higher mRNA and protein levels in relation to the MDRl/P-gp expression in the ADM cells, as compared to the sensitive MCF-7 and MDA-MB-231 cell lines. Meanwhile, we treated MCF-7 and MDA-MB-231 cells with different concentrations of doxorubicin to detect the effect on P-arrestin 2 and MDR1/P-gp expression. We assumed that β-arrestin 2 could regulate multidrug resistance in human breast cancer. Thus, the silence of β-arrestin 2 may result in restoration of multidrug resistance to some extent, which may improve the poor prognosis and increase the survival rate of the drug resistance patients with breast carcinoma.[Methods]1.We examined the expression of P-arrestin 2 in the breast cancer cell lines MCF-7 and MDA-MB-231, as well as in the multidrug resistant subline MCF-7/ADM by RT-PCR and Western blot.2.We treated MCF-7 and MDA-MB-231 cells with 0,0.2μM,1μM and 5μM doxorubicin respectively for 72h and detected P-arrestin 2 and MDR1 mRNA expression using realtime PCR.3. Clinical breast cancer samples were collected and Immunohistochemical Analysis of P-arrestin 2 and MDR1 were used to find the possible relationship between them.4. The MDR cells were all transfected with pSUPER-siFZD1 and pSUPER-siNotarget respectively. After 48h culture, RT-PCR was performed to detect the expression of P-arrestin 2 and MDR1 mRNA, Western blot was used to analysis the β-arrestin 2 and P-gp protein expression.5. MTS assay was used to assess the effect of β-arrestin 2 silencing on the chemosensitivity of MDR cells to 4 anticancer drugs, doxorubicin, paclitaxel, cisplatin, and 5-Fu.[Results]1. p-arrestin 2 was found to have higher mRNA and protein levels in relation to MDR 1/P-gp expression in ADM cells, compared to the sensitive cell lines MCF-7 and MDA-MB-231.2. P-arrestin 2 and MDR1 increased significantly in response to doxorubicin in MCF-7 and MDA-MB-231 cells, in a dose dependent manner.3. The immunohistochemical expression of β-arrestin 2 was positively correlated with MDR1/p-gp. (P=0.016), which was indicated by the Spearman correlation analysis (r = 0.227, P=0.016)4. Both mRNA and protein level of β-arrestin 2 and MDR1 were with the same tendency of changes in the β-arrestin 2 affected breast cancer cell lines groups compared with the negative control groups.5. MTS assay showed that IC50 values of the four anti-cancer drugs all decreased remarkably after β-arrestin 2 interference and the over-expression groups were on the contrary.[Conclusions]Positive correlation between MDR 1/P-gp and β-arrestin 2 was found in the breast cancer cell lines and clinical tissues. The silencing of P-arrestin 2 decreased MDR1 expression and the over-expression was on the contrary, which indicate that β-arrestin2 may be possible to be identified as a new target for MDR reversing in breast cancer. |
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